Nonalcoholic fatty liver disease (NAFLD) is a common liver disease that affects one-third of the world's population. The first stage of NAFLD is hepatic steatosis, which is characterized by accumulation of fat droplets in the cytoplasm of hepatocytes. Hepatic steatosis further develops into nonalcoholic steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma (HCC). Age and diet/fat intake are risk factors for NAFLD. My laboratory investigates age-associated pathways of NAFLD. We found that two members of the C/EBP family, C/EBP? and C/EBP, as well as the chromatin remodeling protein p300 play a critical role in NAFLD associated with aging. Our findings show that cyclin-dependent kinase 4 (cdk4) phosphorylates C/EBP? at Ser193, triggering formation of the tripartite C/EBP?/-p300 complex. Using four animal models with increased or reduced levels of C/EBP?/-p300 complexes, we found that the C/EBP?/-p300 complex activates expression of five key triglyceride (TG) synthesis enzymes. Triglycerides are the main components of the fat droplets that are accumulated in the hepatocytes of NAFLD patients. This led to our main hypothesis that the development of NAFLD is mediated by up-regulation of the cdk4-C/EBP?-p300- TG pathway and that cdk4 inhibitor-mediated reduction in phosphorylation of C/EBP? at Ser193 will inhibit development of NAFLD. We propose to test this hypothesis using animal models of NAFLD and liver biopsies from patients with NAFLD. Specific Aim 1 will examine if knock-in mice carrying C/EBP?- S193A, a C/EBP? construct that cannot be phosphorylated by cdk4, are resistant to development of NAFLD. We recently generated these C/EBP?-S193A mice. We will examine if the development of NAFLD that we normally observe with age and under long-term high fat diet conditions will be reduced in C/EBP?-S193A mice. Specific Aim 2 will test if the inhibition of cdk4 activity by small molecule drugs will inhibit NAFLD. Wild-type mice will be maintained on either high-fat or low-fat diets and treated with inhibitors of cdk4, such as PD0332991. Activity of the cdk4-C/EBP?-p300-TG pathway and development of NAFLD will be compared between un-treated and treated mice. Specific Aim 3 will determine if the cdk4-C/EBP?-p300-TG synthesis pathway is elevated in patients with NAFLD. Our recent data showed that levels of S193-phosphorylated C/EBP? are increased in mouse models of NAFLD and in NAFLD patients. We will therefore: 1) determine if the expression of cdk4 and its activating partner cyclin D3 are elevated in the livers of patients with NAFLD, 2) determine if levels of C/EBP?-p300 complexes are elevated in NAFLD patient liver samples, and 3) examine if TG synthesis enzyme levels are increased in patients with NAFLD. These proposed studies will provide a basis for the development of a novel cdk4-based therapy for NAFLD. Because the cdk4/6 inhibitor PD033991 is already used in the clinical trials for other diseases, the proposed studies might be quickly translated to treatment of human patients with NAFLD.